CHERYL CONOVER

St. Joseph Hospital (C.J.R.), Bangor, Maine 04401; and The Mayo Clinic (C.C.), Rochester, Minnesota 55905

Received June 23, 1997.
Revision received July 30, 1997.
Accepted August 26, 1997.

GH secretion varies over a lifetime. For example, during puberty, boys can secrete as much as 1.0-1.5 mg/day of GH, whereas healthy elderly men can produce as little as 50 mug/day [] . Several factors regulate GH secretion in the elderly. Age-related changes in body composition, declining sex steroid production, reduced physical fitness, aberrant sleep patterns, and poor nutritional status all suppress GH secretion [] . But increased adiposity is probably the strongest inhibitor of GH secretion. Newer studies reveal that individuals with low total body fat exhibit stronger age-related changes in daily GH secretion than obese subjects [] ,[] . In men, the dominant effect of body fat on GH secretion is evident even after controlling for androgen status. For example, total daily GH secretion is strongly and positively correlated with testosterone levels in lean individuals, but not in obese subjects who show reduced GH secretion across all testosterone concentrations [] . Moreover, it is not only the amount of body fat, but its distribution that modulates GH secretion [] . Use of computerized axial tomography and deconvolution analysis of GH secretory rates have demonstrated that visceral fat is a major regulator of GH secretion [] . Whether this is mediated by leptin or other fat-derived substances has not been elucidated.

A confounding issue in assessing the GH/IGF axis has been the pulsatile nature of GH secretion. Dr. Veldhuis's [] laboratory has been leading the field in this endeavor, and recent studies have shed considerable light on this complex process. In particular, his group has reported that advancing age, changes in testosterone, and increased visceral fat deposition leads to greater quantifiable disorderliness (entrophy) of GH release [] . In addition, gender may be critical. Endogenous estrogen production may also be critical because the negative effects of age, physical inactivity, and visceral fat on serum GH concentrations in men are twice as great as they are in women.

All regulatory factors affecting GH release work through the somatostatin and the GHRH pathway. The newer hexapeptide agonists of the growth hormone releasing peptide (GHRP) family and the nonpeptide analogs of such agonists work by facilitating GHRH action and lead to a marked increased in GH secretion in both the elderly and the obese [] . These agents can produce a sustained stimulation of the GH/IGF-I axis, but in contrast to exogenously administered GH, preserve feedback regulation at the pituitary level as well as maintaining a physiological pulsatile pattern of GH release. However, the utility of these agents in diagnosing GH deficiency (GHD) in the elderly has come under some question. Fornito et al. [] demonstrated wide variability in acute GH responses to GHRH even on repeated testing of the same individual. In addition, GH responses to iv GHRH can overlap the normal range even in classic GHD [] . GH responsiveness to GHRPs may be more reproducible but is still modulated by somatostatinergic tone, which is the major source of variability of GH secretion in the elderly. Pretreatment with arginine (which reduces somatostatin secretion) enhances GH responsiveness to GHRH but within-subject variability in the elderly remains high. Hence, the role

of GH secretagogues as diagnostic tools to determine GH insufficiency in the elderly population appears to be very limited.

On the other hand, the future of GH secretagogues as therapeutic agents remains very bright. Repeated doses of these agents can increase GH secretion and IGF-I serum levels. Recent studies in older individuals have only been of short duration (6 weeks or less). However, the GH response to GHRH or GHRP is modulated by negative feedback inhibition via IGF-I and somatostatin, thereby partially protecting elders against overtreatment. In a 28-day trial with a new oral nonpeptide GHRP MK677, GH and plasma IGF-I rose significantly, with the greatest effect occurring after the morning dose [] . Clearly, the oral activity of the GHRPs offers a distinct practical advantage over the iv analogs.

The relevance of serum IGF-I in the aging process continues to remain a major enigma. With improved technology (including better extraction methods to remove confounding IGF binding proteins (IGFBPs) and new immunoradiometric assays for IGF-I and free IGF-I, the measurement of serum IGF-I is now more accurate and precise. Cross-sectional studies from numerous investigators have established a relatively narrow range of serum IGF-I for both healthy and frail elders (80-130 ng/mL for both men and women) and have reconfirmed a significant decline in IGF-I with age in both men and women [] . However, two conditions often found in elders, generalized malnutrition and protein depletion, significantly lower serum IGF-I and alter circulating IGFBPs [] . In contrast to GHD, malnutrition is associated with a 2- to 3-fold rise in GH production [] . This relative GH resistance occurs both at the receptor and postreceptor level. In addition, depletion of certain micronutrients ( e.g. magnesium, thiamine, and zinc) suppresses serum and tissue IGF-I levels [] ,[] . Zinc depletion is common in catabolic states, and this micronutrient may play a critical role in wound healing and protein synthesis, both of which are often impaired in the elderly. Hence, reduced macro- and micronutrient intake, common conditions in the elderly, can be associated with marked changes in serum IGF-I independent of GH status. These data make the interpretation of a single IGF-I measurement tenuous.

Six IGFBPs have been identified in serum. IGFBP-3, the largest, is also the major carrier protein for IGF-I. IGFBP-3 complexes in the circulation with an acid-labile subunit (ALS) and together form a ternary complex with IGF-I and IGF-II. Both IGFBP-3 and ALS are GH dependent. The other five IGFBPs are of lower molecular weight and can translocate across the capillary membrane. Each of the IGFBPs is important in the regulation of serum IGF-I concentration and bioavailability at the site of target tissues. The function of these proteins needs to be better understood, especially in the context of age-related changes in serum IGF-I. Several studies have documented a decline in IGFBP-3 with age. However, Frost et al. [] showed that serum IGFBP-3 concentrations were normal in healthy elders, but markedly decreased in frail seniors who suffered from acute or chronic illnesses. In uncontrolled diabetes mellitus, trauma, surgery, and burns, IGFBP-3 levels fall precipitously, and there is active proteolysis of the intact BP-3 protein [] . During acute illness, there is also disruption of the ternary IGF complex (ALS, IGFBP-3, and IGF-I), in part because of markedly decreased levels of ALS [] .

Frost et al. [] also reported that IGFBP-1 was markedly increased in older individuals, possibly as a result of insulin resistance. Moreover, the predominant IGFBP-1 isoform found in the serum of elderly individuals was the most phosphorylated peptide, one in which the binding affinity for IGF-I is markedly increased. Mohan and colleagues [] reported an age-associated rise in serum IGFBP-4 and a decline in serum IGBP-5.

In summary, examination of the GH/IGF-I axis in elders is extremely complex and must include consideration of numerous hormonal and nonhormonal factors. A single serum or urine test for GH sufficiency in the elderly is not likely to helpful. However, closer examination of the hypothalamic-pituitary axis, the IGFBPs, and the nutrient status of elders may provide important clues to understanding the process of age-related disability.

The functional significance of changes in the GH/IGF-I axis in the elderly is a continuing challenge. Rudman et al. [] first reported that a rise in serum IGF-I induced by exogenous GH could significantly improve skin texture, lean body mass, and bone mineral density in relatively GH deficient elder men. Several subsequent cross-sectional studies showed that the correlation between musculoskeletal frailty and serum levels of IGF-I was weak at best. In fact, few trials have been able to prove that growth factor therapy in the elderly improves physical performance. To date several intervention trials with GH have been published, but in each study, serum IGF-I levels were brought into "young-normal" ranges, yet functional parameters did not change [] [] [] [] . This would suggest a dissociation between attaining "young" levels of IGF-I and restoration of musculoskeletal function in the elderly.

The role of IGF-I as a potential mediator of anabolic action should not be dismissed based purely on studies with systemically administered rhGH or rhIGF-I. Up-regulation of local IGF-I could occur in response to sex steroids, PTH, or vitamin D treatment [] . In fact, changes in the GH/IGF axis within target tissues are often not reflected in the circulation but could account for the anabolic activity of several hormonal therapies. Muscle and bone marrow are good examples. Rooyackers et al. [] and Brodsky et al. [] recently demonstrated an age-associated decline in mitochondrial protein synthesis, which was reversible by testosterone administration at least in hypogonadal men. Because myofibrillar protein synthesis is directly related to IGF-I, it is possible that testosterone administration can increase muscle IGF-I, thereby affecting changes in function. In a similar vein, Glowacki [] demonstrated that in human bone marrow cells extracted at the time of hip surgery in elderly individuals, there were age-associated increases in IGFBP-3. Although these changes are opposite of those seen in the circulation, up-regulation of IGFBP-3 in human bone marrow cells is consistent with other reports that senescent fibroblasts secrete higher amounts of IGFBP-3 [] .

In summary, there are complex changes in tissue-specific IGF regulatory units that make interpretation of any GH/IGF-I intervention difficult at best. Based on the published studies and on work presented at this NIA meeting, most investigators felt that more information was required before concluding that there was a direct relationship between changes in the GH/IGF axis and the functional alterations of aging.

It has long been appreciated that the aging organism is very susceptible to neoplastic transformation and the development of malignancies. The IGF-1 receptor (IGF-IR) is an important component of tumor growth because it both stimulates cell proliferation and protects the cell from apoptosis. Dr. Renata Baserga [] presented data that showed that functional impairment of the IGF-IR achieves the dual goal of inhibiting cell proliferation and inducing massive apoptosis. In particular, targeted disruption of the IGF-IR gene produces a cell refractory to transformation by cellular and viral oncogenes [] . Also, the transformed phenotype can be reversed to a nontransformed phenotype in a number of tumor cells by decreasing the number of IGF-IRs or by interfering with its function. In addition, overexpression of IGF-IR inhibits apoptosis induced by tumor necrosis factor, interleukin-3 withdrawal, etoposide, or other agents [] ,[] . But, two major substrates of the IGF-IR, IRS-I and Shc, do not appear to have protective effects against apoptosis when overexpressed [] . On the other hand, a decrease in IGF-IR levels below normal or the use of dominant negative mutations of the IGF-IR causes massive apoptosis of tumor cells in vivo. Also, interference with the function of the IGF-IR results in tumor cell death, inhibition of tumorigenesis, and prevention of metastases in experimental animals [] . By targeted disruption of the IGF-IR with antisense or dominant negatives, Prisco et al. [] , Sell et al. ([] , and D'Ambrosio et al. [] showed that host cells become totally resistant to subsequent challenges with wild-type C6 tumor cells. In contrast to these works, Plymate and colleagues [] provided preliminary evidence that in human prostatic epithelial cells, as well as in SV40-T immortalized prostate cells, benign to malignant transformation is associated with a decrease in IGF-IR number. Reexpression of the IGF-IR in a highly tumorigenic M12 subline led to a decrease in colony formation and a decrease of in vivo tumor growth and metastases. Despite conflicting data, several lines of evidence suggest that the IGF-IR plays a major role in programmed cell death and may be important in terms of cell responsiveness to challenges incurred by the spread of tumor cells.

Postreceptor signaling mechanisms for the IGF receptor are currently under investigation. The two major signaling cascades for IGF-IR are the Ras/Raf/mitogen-activated protein kinase and the phosphatidyl inositol 3' kinase pathways [] . Using a pheochromocytoma PC12 cell line that undergoes apoptosis following removal of serum or nerve growth factor, Dr. LeRoith [] reported that expression of a dominant negative MAPK/Erk kinase protein abrogated the effect of IGF-I on inhibiting apoptosis. Blockade of the phosphatidyl inositol 3' kinase pathways using a dominant negative p65 molecule stably transfected also resulted in diminution of IGF-I's protective effect against apoptosis [] . Finally, Parrizas and LeRoith [] showed that IGF-I induces the expression of a critical antiapoptotic protein, bcl-xl, in a time- and dose-dependent manner.

p53 is a critical response protein to cellular and genotoxic stress and is essential in safeguarding the replication and propagation of intact chromosomal DNA. A seminal finding by Buckbinder colleagues [] , that one of the target genes for p53 is IGFBP-3, has provided a critical link between p53 and the IGF-I regulatory system. It is conceivable that activation of IGFBP-3 expression by p53 will abrogate survival signals rather than growth signaling from the IGF-IR. Because two distinct p53 response elements in the first and second intron of the IGFBP-3 gene closely match those of the p53 consensus binding site, it is likely that p53 sensitizes cells to apoptosis by interfering with the survival functions of IGFs via induction of IGFBP-3.

The activity of the IGFBPs independent of IGF-I and the release of IGFs from their binding protein are also critical elements of the IGF regulatory system. It is now recognized that IGFBP-3 binds specifically and with high affinity to the surface of various cell types and directly inhibits monolayer cell growth in an IGF-I-independent manner, presumably through interaction with cell membrane proteins that function as an IGFBP-3 receptor [] . At the NIA meeting, Dr. Oh [] reported that a new IGFBP, IGFBP-7, constitutes a low-affinity member of the IGFBPs, with actions similar to those observed with IGFBP-3 in breast cancer cells.

In respect to the liberation of IGFs from their IGFBPs, several IGFBP proteases have been recently identified. Work by Nunn et al. [] and others has led to the characterization of several prostatic proteases including PSA and hK-2 that act on IGFBP-3 and IGFBP-5, including cathepsin D, which is responsible for acid-activated IGFBP proteolysis in seminal plasma, and urokinase, which regulates IGFBP action in prostatic cell lines. It has been suggested that the IGFBPs may serve a dual role, liberating IGFs from the IGFBPs and generating modified IGFBPs with intrinsic IGF-independent activity [] . Once again, these studies reinforce the complexity of the IGF system and its regulatory circuits.

This symposium provided a forum for clinical and basic investigators to discuss salient issues surrounding changes in the GH/IGF axis with aging. Although more work will be needed before any consensus can be reached about the role of the IGF regulatory system in aging and the benefits and limitations of growth factor therapy for the elderly, the framework for evaluating the GH/IGF axis in aging has been established.

Special thanks go to Dr. Stan Slater of the National Institutes of Aging who initially suggested this type of program and constantly provided the necessary assistance to complete the project. In addition, the organizers wish to thank Barbara Kershner for manuscript preparation and coordination of the symposium. These other organizations generously

provided corporate support: Diagnostics Systems Laboratory; Genentech, Inc.; Pfizer Central Research; Merck Laboratories; and Celtrix, Inc.

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